Throughout this application, various publications are referenced by within parentheses. Full citations for these publications may be found at the end of each series of experiments. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed therein.
Malignant melanoma is increasing at a rapid rate in North American populations and it is estimated that 1 in 100 children currently born may eventually develop superficial spreading-type melanoma (1). Although readily curable at early stages, surgical and chemotherapeutic intervention are virtually ineffective in preventing metastatic disease and death in patients with advanced states of malignant melanoma (1). These observations emphasize the need for improved therapeutic approaches to more efficaciously treat metastatic melanoma. A potentially useful therapeutic modality for this and other malignancies could involve the use of agents capable of inducing an irreversible loss in proliferative capacity in tumor cells without the requirement for direct cytotoxicity, that is, the differentiation therapy of cancer (2-5). In previous studies, applicants have demonstrated that it is possible to reprogram human melanoma cells to undergo terminal cell differentiation with a concomitant loss of proliferative capacity by treatment with the combination of recombinant human fibroblast interferon (IFN-β) plus the antileukemic compound mezerein (MEZ) (6,7). The combination of IFN-β+MEZ induces terminal differentiation in melanoma cells, innately resistant to the antiproliferative effect of either agent alone, and in human melanoma cells selected for resistance to growth suppression induced by IFN-β (6,7). In contrast, treatment with IFN-β or MEZ alone results in the development of specific components of the differentiation program in human melanoma cells, but these agents do not induce most melanoma cells to undergo terminal cell differentiation (6-8).
Terminal differentiation induced by IFN-β plus MEZ in human melanoma cells is associated with an increase in melanin synthesis, changes in cellular morphology (characterized by the production of dendrite-like processes), modifications in cell surface antigenic profile, and an irreversible loss of proliferative capacity (3, 6-10). When used separately, IFN-β and MEZ induce both growth suppression and increased melanin synthesis and MEZ induces the production of dendrite-like processes in specific human melanoma cells (6,8). Trans-retinoic acid (RA) is effective in inducing tyrosinase activity and enhancing melanin synthesis in specific human melanoma cultures without altering cell growth, whereas mycophenolic acid (MPA) can induce growth suppression, increased tyrosinase activity and melanin synthesis, and dendrite formation (11). In contrast, the combination of IFN-β+recombinant immune interferon (IFN-γ results in a synergistic suppression in the growth of human melanoma cells without inducing enhanced melanin synthesis or morphologic changes (10,12). These observations suggest that the various changes induced during the process of differentiation in human melanoma cells, that is, increased tyrosinase activity and melanin synthesis, antigenic changes, dendrite formation, and growth suppression, can occur with and without the induction of terminal cell differentiation.
An unresolved issue is the nature of the gene expression changes that occur in human melanoma cells reversibly committed to differentiation vs. human melanoma cells irreversibly committed to terminal differentiation. This information will be important in defining on a molecular level the critical gene regulatory pathways involved in growth and differentiation in human melanoma cells. To begin to address these questions, applicants have used various experimental protocols that result in either growth suppression without the induction of differentiation-associated properties, a reversible induction of differentiation-associated traits, or terminal cell differentiation in the H0-1 human melanoma cell line. As potential target genes relevant to these processes, applicants have analyzed early growth response, extracellular matrix, extracellular matrix receptor, and interferon-responsive genes. No unique gene expression change was observed solely in H0-1 cells induced to terminally differentiate vs. cultures reversibly growth arrested. However, treatment of H0-1 cells with IFN-β+MEZ was associated with specific patterns of gene expression changes that were also apparent in H0-1 cells cultured in conditioned medium obtained from terminal-differentiation-inducer-treated H0-1 cells. Exposure to either the terminal differentiation-inducing compounds or -conditioned medium resulted in the enhanced expression of HLA Class I antigen, melanoma growth stimulatory activity (gro-MGSA), interferon-stimulated gene-15 (ISG-15), and fibronectin. These observations support the potential involvement of a type I interferon and a gro/MGSA autocrine loop in the chemical induction of differentiation in H0-1 cells